Nanocontact printing

ABSTRACT

A method of stamping of molecular patterns and/or devices based on the reversible self-assembly of molecules, particularly organic molecules is disclosed. This method is suitable for the stamping of almost any nanofabricated device and can be used to transferring a large amount of pattern information from one substrate to another at the same time.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Ser. No. 10/688,867, filedon Oct. 17, 2003, which is incorporated in their entirety herein byreference.

GOVERNMENT INTEREST STATEMENT

This invention was made in whole or in part with government supportunder Grant Number DMI-0303821, awarded by the National ScienceFoundation. The government may have certain rights in the invention.

BACKGROUND

In recent years, there has been considerable effort aimed atunderstanding new phenomena in the nanoscale, a diversity of newnanostructured materials have been fabricated and characterized. Newdevices with intriguing properties are just beginning to be engineered.The expectations for a new generation of cheap and innovative tools thatwill change our lives are very high. The combination of a new set ofexpected and unexpected properties together with a whole new family ofmaterials and fabrication methods will enable devices that we could noteven have conceived just ten years ago. Coulomb blockade in metalnanoparticles as well as in semiconductor quantum dots, narrow bandfluorescence emission from semiconductor nanoparticles, quantizedballistic conduction in nanowires and nanotubes: these are just a fewnew materials/phenomena that will have an impact on the way we designoptical and electronics devices. For a review of nanodevices andfabrication techniques, see Bashir, Superlattice and Microstructures(2001), 29(1):1-16; Xia, et al., Chem. Rev. (1999), 99:1823-1848; andGonsalves, et al., Advanced Materials (2001),13(10):703-714, the entireteachings of which are incorporated herein by reference.

Nanoscience development, similarly to many other branches of science,but probably in a more extreme way, relies on state-of the-arttechniques for the imaging and fabrication of its tools. Undoubtedly,the development of the transmission electron microscope (TEM) andscanning tunneling microscope (STM) gave birth to the whole field ofnanoscience. While the development of electron-beam (e-beam)lithography, being the first tool that could build structure and devicesin the nanometer scale, made the field of nanotechnology a reality.

The first stages of nanoscience and mainly of nanotechnology have beendominated by the development and characterization of new materials anddevices based on inorganic semiconductors and metals. One of the mainreasons for this is that e-beam lithography is a technique to patterninorganic materials on an inorganic substrate. A significant advancementin recent years has been the development of novel highly versatilenanolithographies based on scanning probe microscopes (SPM). Usingvarious types of SPMs a wide variety of organic and inorganic substratescan now be patterned either by inducing localized chemical modificationsor by forming self-assembled monolayers (SAMs). For example, Mirkin andcoworkers have developed an atomic force microscope (AFM)-basedtechnique (Dip Pen Nanolithography, DPN) in which a SAM can be generatedby controlled transfer of molecules from the microscope tip to asubstrate, with resolution below 5 nm (see Lee, et al., Science (2002),295:1702-1705; Demers, et al., Angew. Chem. Int. Ed. (2001),40(16):3069-3071; Hong, et al., Science (1999), 286:523-525; Piner, etal., Science (1999), 283:661-663; Demers, et al., Angew. Chem. Int. Ed.(2001), 40(16):3071-3073; Demers, et al., Science (2002), 296:1836-1838,U.S. Patent Application Publication Nos. 2002/0063212, 2003/0049381,2003/0068446, and 2003/0157254, the entire teachings of which areincorporated herein by reference). The development of such techniquesrepresents a major breakthrough, as now it is possible to build devicesbased not only on inorganic but also on organic and biomaterials.Organic based nanomaterials are likely to offer a number of interestingproperties that can be effectively modulated on the nanoscale.Furthermore, the typical disadvantages of organic materials are lessimportant in nanodevices; for example, there is less need for goodmechanical properties or high thermal stability. Thanks both to thesenovel fabrication techniques and to the elucidation of basic concepts insurface and supra-molecular chemistry, novel devices are currently wellunder development.

Using organic and inorganic based nano-lithography techniques manydifferent nano-devices (e.g nano-transistors, nano-sensors andnano-waveguides) are presently being fabricated. However, in order topredict how great an impact nanotechnology will have, one must estimatethe speed of fabrication for complex devices. Unfortunately, allnano-lithographies have in common the same drawback: they are extremelyslow, and it has been postulated that device fabrication time (andreproducibility) will be the main limiting factor in nanotechnology. Inparticular, the problem of how to scale up production has not beensolved. Addressing the problem of production scale-up is critical if wehope to see the enormous amount of knowledge that we are now acquiringtranslated into transistors, sensors, antennas, lenses and drug deliverysystems to use in everyday life.

It would be desirable for nanotechnology to have an equivalent ofmicro-contact printing: this stamping technique engineered by Whitesidesand coworkers (see U.S. Pat. Nos. 5,512,131, 5,900,160, 6,048,623,6,180,239, 6,322,979, 6,518,168, the entire teachings of which areincorporated herein by reference) has revolutionized the way peopledesign micro-devices and has had an enormous impact in allowingnon-chemist to build devices as complex as bio-MEMS. Unfortunately,micro-contact printing has serious resolution limitations, so itsapplication in nanotechnology is limited.

The only research efforts to directly address this problem is that byChou and coworkers at Princeton. In a recent Hewlett Packard pressrelease (“breakthrough in nano-electronics”), their patents and patentapplications on nano-imprinting (U.S. Pat. Nos. 5,772,905 and 6,309,580,and U.S. Patent Application Publication Nos. 2002/0167117, 2003/0034329,2003/0080471, and 2003/0080472, the entire teachings of which areincorporated herein by reference) were considered one of the fundamentalsteps towards the realization of nano-transistors. The method is basedon a hard mold (i.e., a mold made of an inorganic material) that isstamped on a soft polymer film overcoating a silicon wafer. The printedsubstrates typically consist of metallic wires or semiconductormaterials (see Chou, et al., Nature (2002), 417:835-837; and Austin, etal., J. Vac. Sci. Technol. B (2002), 20(2):665-667, the entire teachingsof which are incorporated herein by reference). As with many otherfundamental aspects of nanotechnology, the fabrication methods forinorganic materials are preceding those for organic materials. In fact,the main limitations to nano-imprint is that it needs a “hard” mold andthat it is tailor-made to print a shape on a silicon wafer. It isdifficult to envision how such a method could be adapted for soft molds(i.e., a mold made of an organic material) and/or how it could be usedto transfer the high degree of complexity and information that anorganic (particularly a bio-organic) substrate can carry.

A major drawback of existing nanolithography techniques for fabricatingnanoscale devices is that features of the device must be fabricated in aseries of steps. Thus, these techniques are limited to relatively simpledevices since the fabrication of devices having many features would takea prohibitive amount of time. The only major effort to address thisproblem is the fabrication of multi-tip arrays for SPMs (Zhang, et al.,Nanotechnology (2002), 13:212, the entire teachings of which areincorporated herein by reference). While such approaches will mainlyenable the parallel fabrication of a perhaps tens or hundreds ofnano-devices, it would be desirable to develop a nanoscale stampingtechnique that could complement such parallel device production, andmove it toward mass-production by developing a method that can producemany features in a parallel manner on a device in a single processingstep.

SUMMARY OF THE INVENTION

The method of the invention allows multiple features of a nanoscaledevices to be fabricated at the same time. In one aspect of theinvention, the method involves forming a complement image of a master.The master used in this embodiment of the method of the inventioncomprises a first set of molecules bound to a first substrate to form apattern. The master is contacted with a second set of molecules thatassembling via attractive forces or via bond formation on the first setof molecules. Each molecule in the second set of molecules comprises areactive functional group and a recognition component that is attractedto or binds to at least a portion of one or more of the first set ofmolecules. The reactive functional group of the second set of moleculesis then contacted with a surface of a second substrate. The surface ofthe second substrate reacts with the reactive functional group of thesecond set of molecules to form a bond between the second set ofmolecules and the second substrate. The attractive force between thefirst set of molecules and the second set of molecules is then broken,and the second set of molecules bound to the second substrate forms acomplement image of the master. Once the master has been separated fromthe complement image by breaking the bonds between the first and thesecond set of molecules, the master can be reused one or more times toform additional complement images.

In another aspect of the invention, the method involves forming areproduction of a master, or a portion thereof. The master used in thisembodiment of the method of the invention comprises a first set ofmolecules bound to a first substrate to form a pattern. A second set ofmolecules is assembled on the first set of molecules via bond formation.The second set of molecules comprises a reactive functional group and arecognition component that binds to at least a portion of one or moremolecules from the first set of molecules. The reactive functional groupof the second set of molecules is then contacted with a surface of asecond substrate. The reactive functional group reacts with the surfaceof the second substrate to form a bond between the second set ofmolecules and the second substrate. The bonds between the first set ofmolecules and the second set of molecules are then broken, and thesecond set of molecules bound to the second substrate forms a complementimage of the master. A third set of molecules is then assembled via bondformation on the second set of molecules of the complement image. Eachmolecule in the third set of molecules comprises a reactive functionalgroup, and a recognition component that binds to the second set ofmolecules. The reactive functional group of the third set of moleculesis then contacted with a surface of a third substrate. The surface ofthe third substrate reacts with the reactive functional group of thethird set of molecules to form a bond between the third set of moleculesand the third substrate. The bonds between the second set of moleculesand the third set of molecules are then broken, and the third set ofmolecules bound to the third substrate form a reproduction of thepattern, or portion thereof, of the master. Once the complement imagehas been separated from the reproduction, the complement image can bereused one or more times to form additional reproductions.

In another aspect, the invention relates to a composition comprising amaster having a pattern of a first set of molecules bound to a firstsubstrate; and a complement image comprising a pattern of a second setof molecules bound to a second substrate via a reactive functional groupon each molecule of the second set of molecules. In this aspect of theinvention, each molecule in the second set of molecules has arecognition component that binds to at least a portion of a moleculefrom the first set of molecule.

In another aspect, the invention relates to a composition, comprising afirst pattern of a first set of molecules bound to a first substrate;and a second pattern of a second set of molecules bound to a secondsubstrate via a reactive functional group on each molecule of the secondset of molecules. In this aspect of the invention, each molecule of thesecond set of molecules comprises a recognition component that binds toat least one molecule in the first set of molecules, and the secondpattern is a complement image of the first pattern.

In another aspect, the invention relates to a composition, comprising afirst pattern of a first set of molecules bound to a first substrate;and a second substrate, wherein the second substrate comprises adegraded portion and an undegraded portion, and the undegraded portionis a complement image of the first pattern.

In another aspect, the invention relates to a composition, comprising afirst pattern of a first set of molecules bound to a first substrate;and a second substrate having a patterned layer of a material depositedthereon, wherein the patterned layer of deposited material on the secondsubstrate is a complement image of the first pattern.

In another aspect, the invention relates to a kit for printing amolecular pattern on a substrate. The kit comprises a master comprisinga pattern of a first set of molecules bound to a substrate; and a secondset of molecules. The second set of molecules comprises a reactivefunctional group; and a recognition component that binds to the firstset of molecules.

In another aspect, the invention relates to a molecular printer forgenerating a complement image of a master, wherein the master has afirst set of molecules bound to a first substrate. The molecular printercomprises a device for delivering a solution of a second set ofmolecules to a surface of the master, and a device for contacting thesecond set of molecules with a second substrate. In this embodiment, thesecond set of molecules comprises a reactive functional group; and arecognition component that binds to the first set of molecules.

The method of the invention complements all the chemically orientednanolithography techniques that have been developed in recent years andwhich generally require complex instrumentation. For example, it hasalready been shown that DNA testing arrays can be fabricated using DipPen Nanolithography. Once the master for these devices is built, themethod of the invention, that does not require complex instrumentationand materials, can be used to print a large number of cheap andextremely sensitive devices for the detection of, for example,biohazards. By way of example, let's assume that such a device covers anarea of 1 mm². From a single mold built on a silicon wafer (˜10⁶ mm²),one million sensors for a specific bio-molecule (such as anthrax) couldbe fabricated in approximately 3 hours using the method of theinvention. Because the transfer process is self-assembly based, all thesteps, besides the fabrication of the master, can be done in parallelover very large areas and on multiple substrates.

The amount of information stored in a molecule, such as a DNA strand,can be enormous. The method of the invention has the possibility oftransferring this information in a massively parallel way (i.e., in oneor only a few step instead of many steps). Thus devices that are nowbuilt using multi-step techniques could be fabricated in a single step.This opportunity will redirect research and device manufacture towardsincreasing complexity in fabricated substrates. As a simple example, ifa master were fabricated on a 1 mm² substrate having a series of nanoand microfluidic channels (e.g., 50) having 50 different types of DNAstrands defining the walls of the channels, in one single printing stepwith the method of the invention, one could fabricate on a 1 mm²substrate a complement image of the series of nano and microfluidicchannels, each with the wall functionalized in a different way: a reallab on a chip. This is not possible with any current fabrication method,which would require 50 consecutive steps.

A unique feature of the method of the invention is the ability to copy,and thus replicate, the master itself using the parallel method of theinvention instead of the methods of the prior art in which printed itemscannot act as a master. This is a major advantage over any existingmethods. In fact, typically for large production lines many masters areneeded. This, combined with the wearing of existing molds, means that aconstant production of masters is required. In the method of theinvention, once a master is produced, reproductions of the master can beproduced from it, and these new master will then be used to print thefinal devices. Reproducibility should be improved and, more importantlythe instruments for the primary master fabrication which producefeatures in a serial fashion will have to be used only to fabricate theprimary master.

The method of the invention is revolutionary not only because it can beused to print organic SAMs, but because the method can be used totransfer multiple types of information (e.g., chemical+shape) and toreproduce a master in a parallel fashion.

BRIEF DESCRIPTION OF THE DRAWING

The invention is described with reference to a particular embodimentshown in the figures. The embodiment in the figures is shown by way ofexample and is not meant to be limiting in any way.

FIGS. 1A-D are a schematic representation of one embodiment of themethod of the invention for producing a complement image.

FIG. 2 is a schematic representation of a first set of molecules boundto a second set of molecules.

FIGS. 3A and 3B are AFM images of a master having a monolayer of nucleicacid molecules bound to the surface of a substrate.

FIG. 3C is an AFM image of a complement image of the master shown inFIG. 3A.

FIG. 3D is an AFM image of a complement image of the master shown inFIG. 3B.

FIG. 4A is an AFM image of a master having nucleic acids bound to asubstrate in a grid pattern.

FIG. 4B is an AFM image of a complement image of the master shown inFIG. 4A.

DEFINITIONS

A “master,” as used herein, is a substrate that has a first set ofmolecules bound to a surface of the substrate in a random or non-randompattern. Preferably, the first set of molecules are bound to the masterin a non-random pattern. The first set of molecules may include one ormore different molecules. The information encoded in the pattern may befrom the position of each of the molecules on the surface of thesubstrate and/or the chemical nature of the molecule (e.g., a moleculefrom the first set of molecules having a particular nucleic acidsequence will bind specifically to a nucleic acid molecule having acomplementary sequence).

A “complement image of a master,” as used herein, is an image on asubstrate that is a mirror image, when the pattern on the master isasymmetrical, or a copy, when the pattern on the master is symmetrical,of the spatial and/or chemical information encoded in the master, or aportion thereof. In one embodiment, the complement image is formed bybinding a second set of molecules to a second substrate. For example, ifthe first set of molecules bound to the master are nucleic acidmolecules that form a non-centrosymmetric pattern, a complement image ofthe master will be a mirror image of the master formed on a secondsubstrate with a second set of molecules that are nucleic acids thathave a sequence that is complementary to at least a portion of a nucleicacid sequence from the first set of molecules. Typically, the chemicalinformation transferred to the complement image is not identical to theinformation on the master but is enough information to allow at least aportion of the information from the master to be reproduced. Forexample, when the first and second sets of molecules are nucleic acidmolecules, at least three or more consecutive bases of a molecule fromthe first set of molecules must be complementary three or moreconsecutive bases from the second set of molecules. A complement imagecan be formed from a portion of the pattern on the master by selectingmolecules for the second set of molecules that only bind to a portion ofthe molecules of the first set of molecules that are bound to themaster. When the second set of molecules binds only to a portion of thefirst set of molecule, the height profile of the complement image mayhave two or more levels. In addition, a complement image may encode onlya mirror image of the spatial information encoded in the master or mayencode both the chemical and spatial information encoded in the master.For example, if the first set of molecules bound to the master arenucleic acid molecules that form an asymmetric pattern, a complementimage of the master will be a mirror image of the master formed on asecond substrate with a second set of molecules that are nucleic acidsthat have a sequence that is complementary to at least a portion of anucleic acid sequence from the first set of molecules. In this example,both spatial and chemical information are transferred from the master tothe complement image. Furthermore, only a portion of the chemicalinformation may be transferred to the complement image. For example,when the first set of molecules on the master are nucleic acidmolecules, the second set of molecules that form the complement imagemay be nucleic acid sequences that are complementary to only a portionof a nucleic acid sequence on the master (i.e., is not complementary tothe whole sequence).

A “reproduction of a master,” as used herein, is copy of the spatialand/or chemical information encoded in a pattern of a master. Thereproduction may be a copy of only a portion of the pattern of themaster or may be a copy of the entire pattern of the master. Inaddition, a reproduction of a master may copy only the spatialinformation of the master or may copy both the spatial and chemicalinformation encoded in the master. In addition, a reproduction of amaster may reproduce only part of the chemical information.

“Chemical information encoded in a molecule” refers to the ability ofthe molecule to bind specifically to another molecule or to a specifictype of molecule, typically, in a specific conformation. For example, aparticular nucleic acid sequence binds specifically to a complementarysequence; or protein A binds specifically to immunoglobulins.

The term “pattern,” as used herein, refers to the spatial location ofeach molecule in a set of molecules bound to a substrate, and thechemical structure of each molecule in the set of molecules.

The term “reactive functional group,” as used herein, is a group thatcan react to form a bond with a surface of a substrate. Examples ofreactive functional groups include thiol groups or a protected thiolgroup, which can bind to surfaces made of gold, silver, copper, cadmium,zinc, palladium, platinum, mercury, lead, iron, chromium, manganese,tungsten, or any alloys thereof. Protected thiol groups can bedeprotected before they can bind to the substrate surface. Methods ofprotecting and deprotecting thiol groups can be found in Greene andWuts, “Protective Groups in Organic Synthesis”, John Wiley & Sons(1991), the entire teachings of which are incorporated into thisapplication by reference. Another example of reactive functional groupsis a silane or a chlorosilane, which can bind to a surface of doped orundoped silicon. Another example of a reactive functional group is acarboxylic acid, which can bind to a surface that is an oxide, such assilica, alumina, quartz, or glass. Another example of reactivefunctional groups are nitriles and isonitriles, which can bind to asurface of platinum, palladium or any alloy thereof. Another example ofa reactive functional group is a hydroxamic acid, which can bind to acopper surface.

When a set of molecules binds to the surface of a substrate, themolecules will fold or stack against one another such that a portion ofthe molecule will be exposed on the surface of the substrate. Theexposed functionality may be hydrophobic, hydrophilic, or an amphipathicfunctionality. In addition, the exposed functionality may be afunctionality that selectively binds various biological or otherchemical species such as proteins, antibodies, antigens, sugars andother carbohydrates, and the like. The exposed functionality maycomprise a member of any specific or non-specific binding pair, such aseither member of the following non-limiting list: antibody/antigen,antibody/hapten, enzyme/substrate, enzyme/inhibitor, enzyme/cofactor,binding protein/substrate, carrier protein/substrate,lectin/carbohydrate, receptor/hormone, receptor/effector, complementarystrands of nucleic acid, repressor/inducer, or the like. Examples ofexposed functionalities include —OH, —CONH—, —CONHCO—, —NH₂, —NH—,—COOH, —COOR, —CSNH—, —NO₂, —SO₂, —SH, —RCOR—, —RCSR—, —RSR, —ROR—, —PO₄³, —OSO₃ ⁻², —SO₃ ⁻, —COO⁻, —SOO⁻, —RSOR—, —CONR₂, —(OCH₂CH₂)_(n)OH(where n=1-20, preferably 1-8), —CH₃, —PO₃H⁻, -2-imidazole, —N(CH₃)₂,—N(R)₂, —PO₃H₂, —CN, —(CF₂)_(n)CF₃ (where n=1-20, preferably 1-8), andan olefin. R is hydrogen, a hydrocarbon, a halogenated hydrocarbon, aprotein, an enzyme, a carbohydrates, a lectin, a hormone, a receptor, anantigen, an antibody, or a hapten.

The term “silane,” as used herein, refers to a functional group havingthe following structural formula:

R₂ in the above structural formula, for each occurrence, isindependently selected from the group consisting of —H, an alkyl, anaryl, an alkenyl, an alkynyl, and an arylalkyl.

The term “chlorosilane,” as used herein, refers to a functional grouphaving the following structural formula:

R₆ in the above structural formula, for each occurrence, isindependently selected from —Cl or —OR₂, provided that at least one ofR₆ is —Cl. Preferably, each R₆ is —Cl.

The term “spacer,” as used herein, refers to a divalent group thatconnects two components of a molecule. Preferred spacers includealkylene, a heteroalkylene, a heterocycloalkylene, an alkenylene, analkynylene, an arylene, a heteroarylene, an arylalkylene, and aheteroarylalkylene, wherein the alkylene, heteroalkylene,heterocycloalkylene, alkenylene, alkynylene, arylene, heteroarylene,arylalkylene, or heteroarylalkylene may be substituted or unsubstituted.

The term “alkyl,” as used herein, means a straight chained or branchedC₁-C₂₀ hydrocarbon or a cyclic C₃-C₂₀ hydrocarbon that is completelysaturated. Alkyl groups may be substituted or unsubstituted.

The term “alkylene” refers to an alkyl group that has at least twopoints of attachment to at least two moieties (e.g., methylene,ethylene, isopropylene, etc.). Alkylene groups may be substituted orunsubstituted.

“Alkenyl groups” are straight chained or branched C₂-C₂₀ hydrocarbon ora cyclic C₃-C₂₀ hydrocarbon that have one or more double bonds. Alkenylgroups may be substituted or unsubstituted.

An “alkenylene” refers to an alkenyl group that has two points ofattachment to at least two moieties. Alkenylene groups may besubstituted or unsubstituted.

“Alkynyl groups” are straight chained or branched C₂-C₂₀ hydrocarbon ora cyclic C₃-C₂₀ hydrocarbon that have one or more triple bonds. Alkynylgroups may be substituted or unsubstituted.

An “alkynylene” refers to an alkynyl group that has two points ofattachment to at least two moieties. Alkynylene groups may besubstituted or unsubstituted.

An “heteroalkylene” refers to a group having the formula—X-{(alkylene)-X}_(q)—, wherein X is —O—, —NR₁—, or —S—; and q is aninteger form 1 to 10. R₁ is a hydrogen, alkyl, aryl, arylalkyl, alkenyl,alkynyl, heteroaryl, heteroarylalkyl, or heterocycloalkyl.Heteroalkylene groups may be substituted or unsubstituted.

The term “aryl,” as used herein, either alone or as part of anothermoiety (e.g., arylalkyl, etc.), refers to carbocyclic aromatic groupssuch as phenyl. Aryl groups also include fused polycyclic aromatic ringsystems in which a carbocyclic aromatic ring is fused to anothercarbocyclic aromatic ring (e.g., 1-naphthyl, 2-naphthyl, 1-anthracyl,2-anthracyl, etc.) or in which a carbocylic aromatic ring is fused toone or more carbocyclic non-aromatic rings (e.g., tetrahydronaphthylene,indan, etc.). The point of attachment of an arylene fused to acarbocyclic, non-aromatic ring may be on either the aromatic,non-aromatic ring. Aryl groups may be substituted or unsubstituted.

An “arylene” refers to an aryl group that has at least two points ofattachment to at least two moieties (e.g., phenylene, etc.). Arylenegroups may be substituted or unsubstituted.

An “arylalkyl” group refers to an aryl group that is attached to anothermoiety via an alkylene linker. Arylalkyl groups may be substituted orunsubstituted. When an arylalkylene is substituted, the substituents maybe on either the aromatic ring or the alkylene portion of the arylalkyl.

An “arylalkylene group,” as used herein, refers to an arylalkyl groupthat has at least two points of attachment to at least two moieties. Thesecond points of attachment can be on either the aromatic ring or thealkylene. An arylalkylene may be substituted or unsubstituted. When anarylalkylene is substituted, the substituents may be on either thearomatic ring or the alkylene portion of the arylalkylene.

The term “heteroaryl,” as used herein, means an aromatic heterocyclewhich contains 1, 2, 3 or 4 heteroatoms selected from nitrogen, sulfuror oxygen. A heteroaryl may be fused to one or two rings, such as acycloalkyl, a heterocycloalkyl, an aryl, or a heteroaryl. The point ofattachment of a heteroaryl to a molecule may be on the heteroaryl,cycloalkyl, heterocycloalkyl or aryl ring, and the heteroaryl group maybe attached through carbon or a heteroatom. Examples of heteroarylgroups include imidazolyl, furyl, pyrrolyl, thienyl, oxazolyl,thiazolyl, isoxazolyl, thiadiazolyl, oxadiazolyl, pyridinyl, pyrimidyl,pyrazinyl, pyridazinyl, quinolyl, isoquniolyl, indazolyl, benzoxazolyl,benzofuryl, benzothiazolyl, indolizinyl, imidazopyridinyl, pyrazolyl,triazolyl, isothiazolyl, oxazolyl, tetrazolyl, benzimidazolyl,benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzoxadiazolyl,indolyl, tetrahydroindolyl, azaindolyl, imidazopyridyl, qunizaolinyl,purinyl, pyrrolo[2,3]pyrimidyl, pyrazolo[3,4]pyrimidyl orbenzo(b)thienyl each of which is optionally substituted. Heteroarylgroups may be substituted or unsubstituted.

A “heteroarylene” refers to an heteroaryl group that has at least twopoints of attachment to at least two moieties. Heteroarylene groups maybe substituted or unsubstituted.

A “heteroarylalkyl group” refers to an heteroaryl group that is attachedto another moiety via an alkylene linker. Heteroarylalkyl groups may besubstituted or unsubstituted. When a heteroarylalkylene is substituted,the substituents may be on either the aromatic ring or the alkyleneportion of the heteroarylalkyl. Heteroarylalkyl groups may besubstituted or unsubstituted.

A “heteroarylalkylene” refers to an heteroarylalkyl group that has atleast two points of attachment to at least two moieties.Heteroarylalkylene groups may be substituted or unsubstituted.

A “heterocycloalkyl” refers to a non-aromatic ring which contains one ormore, for example, one to four, oxygen, nitrogen or sulfur (e.g.,morpholine, piperidine, piperazine, pyrrolidine, and thiomorpholine).Heterocycloalkyl groups may be substituted or unsubstituted.

A “heterocycloalkylene” refers to a heterocycloalkyl that has at leasttwo points of attachment to at least two moieties. Heterocycloalkylenegroups may be substituted or unsubstituted.

Suitable substituents for an alkyl, an alkylene, an alkenyl, analkenylene, an alkynyl, an alkynylene, a heteroalkyl, a heteroalkylene,a heterocycloalkyl, a heterocycloalkylene group, an aryl, an arylenegroup, an arylalkyl, an arylalkylene, a heteroaryl, a heteroarylene, aheteroarylalkyl, and a heteroaryalkylene groups include any substituentthat is stable under the reaction conditions used in the method of theinvention. Examples of substituents include an aryl (e.g., phenyl), anarylalkyl (e.g., benzyl), nitro, cyano, halo (e.g., fluorine, chlorineand bromine), alkyl (e.g., methyl, ethyl, isopropyl, cyclohexyl, etc.)haloalkyl (e.g., trifluoromethyl), alkoxy (e.g., methoxy, ethoxy, etc.),hydroxy, —NR₃R₄, —NR₃C(O)R₅, —C(O)NR₃R₄, —C(O)R₃, —C(O)OR₃, —OC(O)R₅,wherein R₃ and R₄ for each occurrence are, independently, —H, an alkyl,an aryl, or an arylalkyl; and R₅ for each occurrence is, independently,an alkyl, an aryl, or an arylalkyl.

Alkyl, alkylene, heterocycloalkylene groups, and any saturated portionof alkenyl, alkenylene, alkynyl, alkynylene groups, may also besubstituted with ═O and ═S.

When a heteroalkylene, a heterocycloalkyl, a heterocycloalkylene, aheteroaryl, or a heteroarylene group contain a nitrogen atom, it may besubstituted or unsubstituted. When a nitrogen atom in the aromatic ringof a heteroaryl or a heteroarylene group has a substituent, the nitrogenmay be a quaternary nitrogen.

The term “nucleic acids,” or “oligonucleotides,” as used herein, refersto a polymer of nucleotides. Typically, a nucleic acid comprises atleast three nucleotides. The polymer may include natural nucleosides(i.e., adenosine, thymidine, guanosine, cytidine, uridine,deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine) ormodified nucleosides. Examples of modified nucleotides include basemodified nucleoside (e.g., aracytidine, inosine, isoguanosine,nebularine, pseudouridine, 2,6-diaminopurine, 2-aminopurine,2-thiothymidine, 3-deaza-5-azacytidine, 2′-deoxyuridine, 3-nitorpyrrole,4-methylindole, 4-thiouridine, 4-thiothymidine, 2-aminoadenosine,2-thiothymidine, 2-thiouridine, 5-bromocytidine, 5-iodouridine, inosine,6-azauridine, 6-chloropurine, 7-deazaadenosine, 7-deazaguanosine,8-azaadenosine, 8-azidoadenosine, benzimidazole, M1-methyladenosine,pyrrolo-pyrimidine, 2-amino-6-chloropurine, 3-methyl adenosine,5-propynylcytidine, 5-propynyluridine, 5-bromouridine, 5-fluorouridine,5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine,8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine), chemically orbiologically modified bases (e.g., methylated bases); modified sugars(e.g., 2′-fluororibose, a minoribose, 2′-azidoribose, 2′-O-methylribose,L-enantiomeric nucleosides arabinose, and hexose), modified phosphategroups (e.g., phosphorpothioates and 5′-N-phosphoramidite linkages), andcombinations thereof. Natural and modified nucleotide monomers for thechemical synthesis of nucleic acids are commercially available.

The term “peptide nucleic acid (PNA),” as used herein, refers to apolymer that has a peptide backbone in which a natural or non-naturalnucleic acid base is attached to each amino acid residue. Peptidenucleic acids are described in Hanvey, et al., Science (1992),258:1481-1485, the entire teachings of which are incorporated byreference. A PNA can bind specifically to a nucleic acid or another PNAthat has a complementary sequence of at least three consecutive bases,preferably six consecutive bases, to the sequence of the PNA.

The term “attractive force,” as used herein, is a force that draws twoor more molecules together. Examples of attractive forces includeattraction of a molecule having a net positive charge to a moleculehaving a net negative charge, dipole-dipole attraction, and magneticattraction.

Unless specified as a covalent bond, the term “bind” or “bound” includesboth covalent and non-covalent associations, such as hydrogen bonds,ionic bonds, covalent bonds, and van der Waals bonds.

The term “recognition component,” as used herein, is a component of amolecule that can bind specifically to another molecule.

“Specific binding,” as used herein, is when a recognition component of amolecule binds one or more other molecule or complex, with specificitysufficient to differentiate between the molecule or complex and othercomponents or contaminants of a sample. Molecules that includerecognition components and their targets are conventional and are notdescribed here in detail. Techniques for preparing and utilizing suchsystems are well known in the art and are exemplified in the publicationof Tijssen, P., “Laboratory Techniques in Biochemistry and MolecularBiology Practice and Theories of Enzyme Immunoassays” (1988), eds.Burdon and Knippenberg, New York: Elsevier, the entire teachings ofwhich are incorporated herein. Preferred recognition components andtheir targets include nucleic acid/complementary nucleic acid,antigen/antibody, antigen/antibody fragment, avidin/biotin,streptavidin/biotin, protein A/I_(g), lectin/carbohydrate andaptamer/target.

As used herein, “aptamer” refers to a non-naturally occurring nucleicacid that binds selectively to a target. The nucleic acid that forms theaptamer may be composed of naturally occurring nucleosides, modifiednucleosides, naturally occurring nucleosides with hydrocarbon linkers(e.g., an alkylene) or a polyether linker (e.g., a PEG linker) insertedbetween one or more nucleosides, modified nucleosides with hydrocarbonor PEG linkers inserted between one or more nucleosides, or acombination of thereof. In one embodiment, nucleotides or modifiednucleotides of the nucleic acid ligand can be replaced with ahydrocarbon linker or a polyether linker provided that the bindingaffinity and selectivity of the nucleic acid ligand is not substantiallyreduced by the substitution (e.g., the dissociation constant of theaptamer for the target should not be greater than about 1×10⁻⁶ M). Thetarget molecule of a aptamer is a three dimensional chemical structurethat binds to the aptamer. However, the aptamer is not simply a linearcomplementary sequence of a nucleic acid target but may include regionsthat bind via complementary Watson-Crick base pairing interruptedby'other structures such as hairpin loops). Targets of aptamers includepeptide, polypeptide, carbohydrate and nucleic acid molecules.

DETAILED DESCRIPTION

The method of the invention involves stamping of molecular patternsand/or devices based on the reversible self-assembly of molecules,particularly organic molecules. This method is suitable for the stampingof almost any nanofabricated device, inorganic and/or organic, and canbe used to transferring a large amount of information from one substrateto another. The working principle of this technique is completelydifferent from any present nanofabrication technique.

In the method of the invention, a master, that includes a substratehaving a first set of molecules bound to at least one surface in apattern, is used to induce the assembly of a second set of molecules viareversible supra-molecular chemistry (e.g., hydrogen bonds, ionic bonds,covalent bonds, van der Waals bonds, or a combination thereof); then,with the use of substantially irreversible surface chemistry, the secondset of molecules are attached to a surface of a substrate andsubsequently the reversible bonds between the first set of molecules andthe second set of molecules are broken. The term “substantiallyirreversible,” as used herein, means that the second set of moleculesare attached to the surface of the substrate by bonds that are stable toconditions that will break the bonds between the first and the secondset of molecules. The method of the invention uses supra-molecular bondsas a means for shape-transfer, this avoids the need for mechanicalcontacts, and thus constitutes a major departure from nano-imprintingdeveloped by Chou and co-workers. This method is tailor-made to transferorganic patterns reliably. The use of organic molecules allows a greatnumber of variations and enables the transfer of multiple surfacefeatures at the same time.

Referring to FIG. 1, in one embodiment, the method of forming acomplement image of a master involves providing a master 10 thatcomprises a first set of molecules 12 bound to a first substrate 14 toform a pattern. A second set of molecules 16 is assembled on the firstset of molecules via bond formation. The second set of moleculescomprises a reactive functional group 18 and a recognition component 20(not shown in FIG. 1) that binds to the first set of molecules 12 (seeFIG. 2 which provides a blowup of the second set of molecules bound tothe first set of molecules). The reactive functional group 18 of thesecond set of molecules 16 is then contacted with a surface of a secondsubstrate 22. The reactive functional group reacts with the surface ofthe second substrate to form a bond between the second set of moleculesand the second substrate. In one embodiment, the remaining exposedsurface of the second substrate may be further contacted with anothergroup of molecules 24 that each have a reactive functional groups, suchas an alkane having a thiol substituent, that can bind to the surface inorder to cover the exposed surface of the second substrate. The bondsbetween the first set of molecules and the second set of molecules arethen broken, and the second set of molecules bound to the secondsubstrate forms a complement image of the master 26. Once the master hasbeen separated from the complement image by breaking the bonds betweenthe first and the second set of molecules, the master can be reused oneor more times to form additional complement images. In one embodiment, alateral dimension of at least one feature of the complement image isless than 200 nm.

In one embodiment, the second set of molecules may also include one ormore of the following components: an exposed functionality 28; acovalent bond or a first spacer 30 that links the reactive functionalgroup to the recognition component; and a covalent bond or a secondspacer that links the exposed functionality to the recognitioncomponent.

The second set of molecules may include two or more different molecules.For example, two or more molecules of the second set of molecules mayhave different recognition components, such as different nucleic acidsequences; or two or more molecules of the second set of molecules mayhave both different recognition components and different exposedfunctionalities. Typically, one or more molecules from the first set ofmolecules determines where each of the molecules from the second set ofmolecules binds.

In one embodiment, the two or more different molecules of the second setof molecules form a pattern on the second substrate that has a heightprofile that comprises two or more depths. For example, two or moremolecules of the second set of molecules may have either a first spacer,a second spacer or both a first and a second spacer that have differentlengths. The difference in the length of the spacers can cause themolecular image transferred to the second substrate to have two or moredifferent depths.

In one embodiment, the second set of molecules is assembled on the firstset of molecules by contacting the master with a solution comprising thesecond set of molecules. In one method of transferring the pattern on amaster to a second substrate, the master is held in contact with thesecond substrate by capillary action of the solution containing thesecond set of molecules. A small mechanical force may also be applied tohold the two substrates together. The solution containing the second setof molecules is then slowly evaporated causing the master and the secondsubstrate to come closer together and facilitating binding of the secondset of molecules to the second substrate.

The bonds formed between the first set of molecules and the second setof molecules may be hydrogen bonds, ionic bonds, covalent bonds, van derWaals bonds, or a combination thereof. Preferably, the bonds formedbetween the first set of molecules and the second set of molecules arehydrogen bonds. In one embodiment, the bonds between the first set ofmolecules and the second set of molecules are broken by applying heat.In another embodiment, the bonds between the first set of molecules andthe second set of molecules are broken by contacting the bonds with asolution having a high ionic strength. In yet another embodiment, thebonds between the first set of molecules and the second set of moleculesare broken by contacting the bonds with a solution having a high ionicstrength and applying heat. Alternatively, the bonds between the firstset of molecules and the second set of molecules are broken bycontacting them with a solution containing an enzyme that breaks thebonds. Typically, the bonds between the first set of molecules and thesecond set of molecules can be broken without breaking most of the bondsbetween the second set of molecules and the second substrate.

The reactive functional group on the second set of molecules istypically a group that can bind to the surface of the second substrate.For example, when the reactive functional group on the second set ofmolecules is a thiol group or a protected thiol group, the surface ofthe second substrate may be gold, silver, copper, cadmium, zinc,palladium, platinum, mercury, lead, iron, chromium, manganese, tungsten,or any alloys thereof. In another example, the reactive functional groupon the second set of molecules is a silane or a chlorosilane, and thesurface of the second substrate is doped or undoped silicon. In anotherexample, the reactive functional group on the second set of molecules isa carboxylic acid, and the surface of the second substrate is an oxide,such as silica, alumina, quartz, or glass. In another example, thereactive functional group on the second set of molecules is a nitrile oran isonitrile, and the surface of the second substrate is platinum,palladium or any alloy thereof. In another example, the reactivefunctional group on the second set of molecules is a hydroxamic acid,and the surface of the second substrate is copper.

In one embodiment, at least some of the molecules of the first set ofmolecules include a recognition component that binds to the one or moremolecules of the second set of molecules. For example, each of themolecules of the first set of molecules may include a recognitioncomponent that is a nucleic acid sequence. In one embodiment, each ofthe first set of molecules is a nucleic acid sequence and therecognition component of the second set of molecules is a nucleic acidsequence. Preferably, the nucleic acid recognition component of each ofthe second set of molecules is complementary to at least a portion of anucleic acid sequence of at least one of the molecules from the firstset of molecules. For example, three or more consecutive nucleic acidbases, preferably six or more nucleic acid bases, of a molecule from thesecond set of molecules is complementary with three or more consecutivenucleic acid bases, preferably six or more nucleic acid bases, of amolecule from the first set of molecules. When the second set ofmolecules is assembled on the first set of molecules, the second set ofmolecules will hybridize with molecules from the first set of moleculesthat have a complementary sequence, or a portion thereof, to the nucleicacid recognition component of the second set of molecules. In thisembodiment, typically, the first set of molecules bound to the masterare contacted with a solution of the second set of molecules underconditions that promote hybridization. Conditions that promotehybridization are known to those skilled in the art. A generaldescription of hybridization conditions are discussed in Ausebel, F. M.,et al., Current Protocols in Molecular Biology, Greene Publishing Assoc.and Wiley-Interscience, 1989, the teachings of which are incorporatedherein by reference. Factors such as sequence length, base composition,percent mismatch between the hybridizing sequences, temperature andionic strength influence the stability of nucleic acid hybrids.

In one embodiment, the first set of molecules includes two or moredifferent molecules that have recognition components that are differentnucleic acid sequences. In this embodiment, the second set of moleculesincludes molecules that have a nucleic acid sequence, or a portionthereof, that is complementary to at least one of the molecules of thefirst set of molecules. In one embodiment, hydrogen bonds betweenhybridized molecules from the first set of molecules and the second setof molecules are broken by contacting the hydrogen bonds with an enzyme.For example, an enzyme from the helicase family of enzymes may be use tobreak the bonds between hybridized nucleic acid molecules. Varioushelicases have been reported to dehybridize double strandedoligonucleotides. For example, E. coli Rep, E. coli DnaB, E. coli UvrD(also known as Helicase II), E. coli RecBCD, E. coli RecQ, bacteriophageT7 DNA helicase, human RECQL series; WRN(RECQ2), BLM(RECQL3), RECQL4,RECQL5, S. Pombe rqh1, C. elegance T04A11.6 (typically, the helicasename is derived from the organism from which enzymes comes). Helicasescan be divided into two types: 1) helicase that move along the nucleicacid strand in the 3′ direction, and 2) helicases that more along thenucleic acid strand in the 5′ direction one. Typically, the particulartype of helicase used to break the hydrogen bonds between the hybridednucleic acids are selected by considering structural hindrance of theparticular hybridized nucleic acids. Cofactors which stabilize singlestranded DNA, such as single stranded DNA binding protein (SSB), couldbe added.

Another method of breaking the bonds between two hybridized nucleicacids would be to use a restriction endonuclease, which recognizesspecific base sequence and cleaves both strands at a specific locationin the nucleic acid sequence. Examples of restriction endonucleasesinclude BamHI, EcoRI, and BstXI. Other methods of dehybridazition ofnucleic acids using enzymes can be found in Lubert Stryer, Biochemistry,4th Edition; Benjamin Lewin, Gene VII; Kristen Moore Picha and Smita S.Patel, “Bacteriophage T7 DNA Helicase Binds dTTP, Forms Hexamers, andBinds DNA in the Absence of Mg2+,” J. Biol. Chem. (1998), Vol. 273,Issue 42, 27315-27319; Sheng. Cui, Raffaella Klima, Alex Ochem, DanieleArosio, Arturo Falaschi, and Alessandro Vindigni, “Characterization ofthe DNA-unwinding Activity of Human RECQ1, a Helicase SpecificallyStimulated by Human. Replication Protein A,” J. Biol. Chem. (2003), Vol.278, Issue 3, 1424-1432; Umezu, K., and Nakayama, H. (1993), J. Mol.Biol., 230:1145-1150; Nakayama, K., Irino, N., and Nakayama, H., Mol.Gen. Genet. (1985), 200:266-271; Kusano, K., Berres, M. E., and Engels,W. R., Genetics (1999), 15:1027-1039; Ozsoy, A. Z., Sekelsky, J. J., andMatson, S. W., Nucleic Acids Res. (2001), 29:2986-299, the entireteachings of these references is incorporated herein by reference.

Alternatively, the bonds between the first set of molecules and thesecond set of molecules are broken by applying heat, by contacting thebonds with a solution having a high ionic strength, or by contacting thebonds with a solution having a high ionic strength and applying heat.

Typically, the nucleic acid sequence of the first and second sets ofmolecules are DNA, RNA, modified nucleic acid sequences or anycombinations thereof.

In an alternative embodiment, a component of each of the first set ofmolecules is a peptide nucleic acid (PNA) sequence and the recognitioncomponent of the second set of molecules is a PNA sequence.Alternatively, a component of each of the molecules from the first setof molecules is a peptide nucleic acid (PNA) sequence and therecognition component of the second set of molecules is a nucleic acidsequence, or vice versa. PNA molecules hybridize to other PNA moleculesand to nucleic acid sequences in a manner similar to that of nucleicacid hybridization to other nucleic acid. Thus, at least one or moremolecule from the second set of molecules must have at least threeconsecutive bases, preferably six consecutive bases, that arecomplementary to three consecutive bases, preferably six consecutivebases, of a molecule from the first set of molecules.

When a set of molecules bind to the surface of a substrate, themolecules will fold or stack against one another such that a portion ofthe molecule will be exposed on the surface of the substrate. Theexposed functionality may be hydrophobic, hydrophilic, or an amphipathicfunctionality. In addition, the exposed functionality may be afunctionality that selectively binds various biological or otherchemical species such as proteins, antibodies, antigens, sugars andother carbohydrates, and the like. Other examples of exposedfunctionalities include —OH, —CONH—, —CONHCO—, —NH₂, —NH—, —COOH, —COOR,—CSNH—, —NO₂ ⁻, —SO₂, —SH, —RCOR—, —RCSR—, —RSR, —ROR—, —PO₄ ⁻³, —OSO₃⁻², —SO₃ ⁻, —COO⁻, —SOO⁻, —RSOR—, —CONR₂, —(OCH₂CH₂)_(n)OH (wheren=1-20, preferably 1-8), —CH₃, —PO₃H⁻, -2-imidazole, —N(CH₃)₂, —N(R)₂,—PO₃H₂, —CN, —(CF₂)_(n)CF₃ (where n=1-20, preferably 1-8), and anolefin, wherein, R is hydrogen, a hydrocarbon, a halogenatedhydrocarbon, a protein, an enzyme, a carbohydrates, a lectin, a hormone,a receptor, an antigen, an antibody, or a hapten.

The exposed functionality may include a protecting group which may beremoved to effect further modification of the complement image or thereproduction of the master. For example, a photoremovable protectinggroup may be used. A wide variety of positive light-reactive groups areknown in the art, for example, nitroaromatic compounds such aso-nitrobenzyl derivatives or benzylsulfonyl. Photoremovable protectivegroups are described in, for example, U.S. Pat. No. 5,143,854, theentire teachings of which are incorporated herein by reference, as wellas an article by Patchornik, JAGS, 92:6333 (1970) and Amit et al, JOC,39:192, (1974), both of which are incorporated herein by reference.

In one embodiment, the complement image can be further modified bybinding the exposed functional group of at least one of the second setof molecules to a metal or a metal ion. For example, when the exposedfunctional group is —SH, the metal or metal ion can be Au°, Ag°, or Ag⁺.Alternatively, when the exposed functional group is —COOH, the metal ormetal ion can be Ag° or Ag⁺.

The second set of molecules may have a first spacer, a second spacer, ora first and the second spacer. The spacers may be, independently,selected from the group consisting of an alkylene, a heteroalkylene, aheterocycloalkylene, an alkenylene, an alkynylene, an arylene, aheteroarylene, arylalkylene, and a heteroarylallylene. The alkylene,heteroalkylene, heterocycloalkylene, alkenylene, alkynylene, arylene,heteroarylene, arylalkylene, and heteroarylalkylene spacers may besubstituted or unsubstituted. In one embodiment, either the first or thesecond spacers, or both the first and the second spacers are substitutedwith one or more halogen and/or hydroxy.

The master may be prepared by any method known to those skilled in theart (see Xia, et al., Chem. Rev. (1999), 99:1823-1848, the entireteachings of which are incorporated by reference). Preferably, themethod of forming the master is a nanopatterning method. In oneembodiment, the master is prepared by forming a pattern of one or moremetal, metal oxide, or combinations thereof on a surface of a substrateusing electron beam lithography. The surface of the substrate is thencontacted with a first set of molecules. In this embodiment, each of thefirst set of molecules has a reactive functional group that forms a bondbetween the metal or metal oxide and the molecules of the first set ofmolecules, so that the first set of molecules binds to the substrateforming a master having a first set of molecules bound to the substrateto form a pattern. For example, when the reactive functional group of atleast one molecule from the first set of molecules is a thiol group or aprotected thiol group, at least a portion of the patterned formed may bea metal selected from the group consisting of gold, silver, copper,cadmium, zinc, palladium, platinum, mercury, lead, iron, chromium,manganese, tungsten, and any alloys thereof. In another example, whenthe reactive functional group of at least one molecule from the firstset of molecules a silane or a chlorosilane, at least a portion of thepatterned formed is a metal selected from the group consisting of dopedand undoped silicon. In another example, when the reactive functionalgroup of at least one molecule from the first set of molecules is acarboxylic acid, at least a portion of the patterned formed is an oxideselected from the group consisting of silica, alumina, quartz, andglass. In another example, when the reactive functional group of atleast one molecule from the first set of molecules is a nitrile or anisonitrile, at least a portion of the patterned formed is a metalselected from the group consisting of platinum, palladium and alloysthereof. In another example, when the reactive functional group of atleast one molecule from the first set of molecules is a hydroxamic acid,at least a portion of the patterned formed is copper.

Alternatively, the master can be prepared using dip pen nanolithography.Methods of preparing molecularly patterned substrates using dip pennanolithography are described in Schwartz, Langmuir (2002), 18:4041-4046and in Piner, et al., Science (1999), 283:661-663, the entire teachingsof both references are incorporated herein by reference.

Alternatively, the master can be prepared using replacement lithography,nanoshading or nanografling. These methods are described in Sun, et al.,JACS (2002), 124(11):2414-2415; Amro, et al., Langmuir (2000),16:3006-3009; Liu, et al., Nano Letters (2002), 2(8):863-867; and Liu,et al., Acc. Chem. Res. (2000), 33:457-466; the entire teachings ofthese references are incorporated herein by reference.

Another embodiment is a lithographic method in which at least oneportion of the second substrate surface is free of the second set ofmolecules. In this embodiment, the exposed surface of the secondsubstrate is contacted with a reactant selected to be chemically inertto the second set of molecules and to degrade at least the surface layerof the second substrate, thereby degrading the portion of the surface ofthe second substrate that is free of the second set of molecules.Typically, the reactant is a reactive ion etching compound. The secondset of molecules is then removed to uncover a portion of the surface ofthe second substrate.

In another embodiment, at least one portion of the second substratesurface is free of the second set of molecules, and a material isdeposited on the portion of the second substrate surface that is free ofthe second set of molecules. Examples of deposited material includesemiconductors, dielectrics, metals, metal oxides, metal nitrides, metalcarbides, and combinations thereof. The second set of molecules is thenremoved to uncover a portion of the surface of the second substrate.

In one aspect of the invention, the method of forming a complement imageof a master involves assembling a second set of molecules via attractiveforces on the first set of molecules. Examples of attractive forcesinclude attraction of a molecule having a net positive charge to amolecule having a net negative charge, dipole-dipole attraction, andmagnetic attraction. In one preferred embodiment, the attractive forceis a magnetic force. In one example, when the attractive force is amagnetic force, one or more molecules from the first set of moleculesand from the second set of molecules include an iron or iron oxidecomponent. In this embodiment, the attractive forces between the firstset of molecules and the second set of molecules can be broken byapplying a magnetic field.

In another aspect of the invention, the method involves forming areproduction of a master, or a portion thereof. The master used in thisembodiment of the method of the invention comprises a first set ofmolecules bound to a first substrate to form a pattern. A second set ofmolecules is assembled on the first set of molecules via bond formation.The second set of molecules comprises a reactive functional group and arecognition component that binds to the first set of molecules. Thereactive functional group of the second set of molecules is thencontacted with a surface of a second substrate. The reactive functionalgroup reacts with the surface of the second substrate to form a bondbetween the second set of molecules and the second substrate. The bondsbetween the first set of molecules and the second set of molecules arethen broken, and the second set of molecules bound to the secondsubstrate forms a complement image of the master. A third set ofmolecules is then assembled via bond formation on the second set ofmolecules of the complement image. Each molecule in the third set ofmolecules comprises a reactive functional group, and a recognitioncomponent that binds to the second set of molecules. The reactivefunctional group of the third set of molecules is then contacted with asurface of a third substrate. The surface of the third substrate reactswith the reactive functional group of the third set of molecules to forma bond between the third set of molecules and the third substrate. Thebonds between the second set of molecules and the third set of moleculesare then broken, and the third set of molecules bound to the thirdsubstrate form a reproduction of the pattern, or portion thereof, of themaster. Once the complement image has been separated form thereproduction, the complement image can be reused one or more times toform additional reproductions. In one embodiment, a lateral dimension ofat least one feature of the reproduction is less than 200 nm.

The method of forming a reproduction is the same as that used to form acomplement image except that the complement image of the master is usedas a template (or “master”) to transfer the pattern to the thirdsubstrates. Thus, the embodiments and examples disclosed above for thesecond set of molecules and the second substrate apply as well to thethird set of molecules and the third substrate, respectively. Inaddition, examples of conditions for assembling the second set ofmolecules on the first set of molecules and for breaking the bondsbetween the first and the second set of molecules can apply equally aswell to conditions for assembling the third set of molecules on thesecond set of molecules and for breaking the bonds between the third andthe second set of molecules.

In another aspect, the invention relates to a composition comprising amaster having a pattern of a first set of molecules bound to a firstsubstrate; and a complement image comprising a pattern of a second setof molecules bound to a second substrate via a reactive functional groupon each molecule of the second set of molecules. In this aspect of theinvention, each molecule in the second set of molecules has arecognition component that binds to at least a portion of a moleculefrom the first set of molecule.

In another aspect, the invention relates to a composition, comprising afirst pattern of a first set of molecules bound to a first substrate;and a second pattern of a second set of molecules bound to a secondsubstrate via a reactive functional group on each molecule of the secondset of molecules. In this aspect of the invention, each molecule of thesecond set of molecules comprises a recognition component that binds toat least one molecule in the first set of molecules, and the secondpattern is a complement image of the first pattern. Alternatively, theinvention relates to a composition, comprising a first pattern of afirst set of molecules bound to a first substrate; and a third patternof a third set of molecules bound to a third substrate via a reactivefunctional group on each molecule of the third set of molecules. In thisaspect of the invention, each molecule of the third set of moleculescomprises a recognition component that binds to at least one molecule inthe second set of molecules, and the third pattern is a reproduction ofthe first pattern, or a portion thereof.

In another aspect, the invention relates to a composition, comprising afirst pattern of a first set of molecules bound to a first substrate;and a second substrate, wherein the second substrate comprises adegraded portion and an undegraded portion, and the undegraded portionis a complement image of the first pattern.

In another aspect, the invention relates to a composition, comprising afirst pattern of a first set of molecules bound to a first substrate;and a second substrate having a patterned layer of a material depositedthereon, wherein the patterned layer of deposited material on the secondsubstrate is a complement image of the first pattern.

In another aspect, the invention relates to a kit for printing amolecular pattern on a substrate. The kit comprise a master comprising apattern of a first set of molecules bound to a substrate; and a secondset of molecules. The second set of molecules comprises a reactivefunctional group; and a recognition component that binds to the firstset of molecules.

In another aspect, the invention relates to a molecular printer forgenerating a complement image of a master, wherein the master has afirst set of molecules bound to a first substrate. The molecular printercomprises comprising a device for delivering a solution of a second setof molecules to a surface of the master, and a device for contacting thesecond set of molecules with a second substrate. In this embodiment, thesecond set of molecules comprises a reactive functional group; and arecognition component that binds to the first set of molecules.

Generally, the apparatus comprises one or more reservoirs that containthe second set of molecules, one or more vessels or components forholding a master in position for delivery of the solution containing thesecond set of molecules. In addition, the apparatus may include acomputer controlled means for transferring in a predetermined manner thesolution of the second set of molecules from the reservoirs to thesurface a master: A clamp that secures the master to the secondsubstrate may also be included in the apparatus of the invention. Thetemperature of the solution of the second set of molecules and thevessel containing the master may also be controlled. The apparatus mayalso include a reservoir containing a solution for breaking the bondsbetween the first and the second molecules, such as a solution having ahigh ionic strength or a solution containing an enzyme that will breakthe bonds, and a means for delivering the solution. In addition, afterthe second substrate has been bound to the second set of molecules, aheating element may be used to heat a solution in contact with the boundfirst and second sets of molecules to break the bonds. The computercontrolled means for transferring solutions and controlling temperaturecan be implemented by a variety of general purpose laboratory robots,such as that disclosed by Harrison et al, Biotechniques, 14: 88-97(1993); Fujita et al, Biotechniques, 9: 584-591 (1990); Wada et al, Rev.Sci. Instrum., 54: 1569-1572 (1983), the entire teachings of thesereferences are incorporated herein by reference. Such laboratory robotsare also available commercially, e.g. Applied Biosystems model 800Catalyst (Foster City, Calif.). In one embodiment, the apparatus alsoincludes a device for separating the second substrate from the masterafter the bonds between the first set of molecules and the second set ofmolecules have been broken.

These and other aspects of the present invention will be furtherappreciated upon consideration of the following Examples, which areintended to illustrate certain particular embodiments of the inventionbut are not intended to limit its scope, as defined by the Claims.

EXAMPLES Example 1 Preparation of a Complement Image of a DNA Monolayer

A. Preparation of DNA Solutions

All glassware was cleaned with a solution of 75% H₂SO₄ and 25% H₂O₂before use. All water used was ultrapure water (18MΩ/cm). The primaryDNA, 5′-/5-ThiolMC6-D/ACG CAA CTT CGG GCT CTT-3′, were purchased fromIntegrated DNA Technologies, Inc. (IDT), Coraville, Iowa. All DNAstrands were used as received from the manufacturer. The primary DNA wasdissolved in water at the concentration of 1 μg/mL and divided intosmaller aliquots of 50 μL, and stored at −20° C. When a portion of thissolution was used, an aliquot was reduced by placing it in a 40 mMbuffer solution (0.17 M sodium phosphate, pH 8) having dithiothreitol(DTT) for 16 hr. The oligonucleotides were separated from theby-products of the DTT reaction using size exclusion chromatography (NAP10 column from Pharmacia Biotech) following the manufacturesinstructions. 10 mM sodium phosphate buffer (pH 6.8) was used toequilibrate the column and to elute the oligonucleotides. Theconcentration of the resulting DNA solution was calculated from theabsorbance of the solution at 260 nm. In the case of primary DNA (i.e.,the DNA used to form the master), 1M potassium phosphate buffer solution(pH 3.8) was added to the DNA solution to increase the ionic strength ofthe solution. The final concentration of DNA was 4-5 μM.

In the case secondary DNA solution (i.e., DNA used to form thecomplement image), 1M NaCl in TE buffer (10 mM Tris buffer pH 7.2 and 1mM EDTA) was added to increase the ionic strength of the solution. Thesecondary DNA used was purchased from Integrated DNA Technologies, Inc.(IDT), Coraville, Iowa and had the following structure5′-/5ThiolMC6-D/AAG AGC CCG AAG TTG CGT-3′.

B. Preparation of a Master Having a DNA Monolayer

Clean and atomically flat gold on mica was used as a substrate. Thissubstrate was placed in the primary DNA solution prepared above for 5days to allow the DNA to bind to the surface of the substrate. Thesubstrate was rinsed with 1M potassium phosphate buffer 2 times and withwater 5 times. The substrate was exposed to 1 mM spacer thiol,6-mercapto-1-hexanol, aqueous solution for 2 hr to minimize nonspecificadsorption of single-stranded DNA, then rinsed with water 5 times.

C. Preparation of Complement Image

The master prepared in step B was dipped into the secondary DNA solutionfor 2 hours to allow the complementary DNA to hybridize to the DNA boundto the master. The substrate was rinsed with 1M NaCl in TE buffer 2times and with water 5 times.

A second clean gold on mica substrate was placed in contact with themaster so that the two gold surfaces were facing each other and had asmall amount of water in between them. A small mechanical force wasapplied to push the two substrates together. As water between twosubstrates was evaporating, the spacing between the surfaces decreaseddue to increasing capillary attraction forces. Consequently, thiolgroups of secondary DNA approached the second substrate and bound to it.After about 5 hr, the substrates were dipped into 1M NaCl in TE buffersolution (70° C.) for 20 mM. The substrates (i.e., the master and thecomplement image) spontaneously separated and were rinsed with 1M NaClin TE buffer 2 times and with water 5 times, then air-dried. Both themaster (see FIGS. 3A and 3B) and the complement image (see FIGS. 3C and3D) were imaged using AFM tapping mode.

D. Results

The coverage of the first substrate surface with DNA was complete. Thethorough coverage made AFM imaging difficult due to strong interactionbetween monolayer and a tip. The layer transferred to the secondsubstrate also had complete coverage.

Example 2 Pattern Transfer of Gold Grid

An AFM calibration gold grid was dipped in 4W solution of the primaryDNA molecules described in Example 1 for 5 days to generate a patternedmaster. The master was exposed to 1 mM 6-mercapto-1-hexanol aqueoussolution for 2 hr to minimize nonspecific adsorption of single-strandedDNA, then rinsed with water 5 times and air-dried. The master was thenexposed to a 6 μM solution of the secondary DNA described in Example 1for 2 hours so that hybridization occurred. A second substrate of goldon mica was placed on the master so that the two gold surfaces werefacing each other and had a small amount of water in between them. Asmall mechanical force was applied to force the two substrates together.After about 5 hr, the substrates were dipped into 1M NaCl in TE buffersolution (70° C.) for 20 min. The two substrates (i.e., master and thecomplement image) spontaneously separated and were rinsed with 1M NaClin TE buffer 2 times and with water 5 times, then air-dried. Both themaster and the complement image were imaged using AFM tapping mode (seeFIGS. 4A and 4B, respectively).

Example 3 Fabrication of a DNA Chip

A master is prepared using Dip Pen Nanolithography, as described inDemer, et al., Angew. Chem. Int. Ed. (2001), 40:30713073, the entireteachings of which are incorporated herein by reference. To prepare themaster, a surface of a gold on mica substrate is contacted with a 1 mMsolution of 1-octadecanethiol (ODT) in ethanol for 5 min. to cover theexposed gold surface with ODT molecules. The substrate is then immersedin a 1 mM solution of 1,16-mercaptohexadecanoic acid (MHA) and the tipof an atomic force microscope is used to displace ODT molecules bound tothe surface by contacting the surface with a force of about 0.5 nNmaking a 100 nm dot. The MHA in solution binds to the exposed goldsurface of the dot. The carboxylic acid groups of the MHA are activatedwith a 10 mg/mL solution of1-ethyl-3-(3-dimethylaminoproppyl)carbodiimide hydrochloride (EDAC) in0.1M morpholine/ethanesulfonic acid at pH 4.5, and then rinsed with asolution of 0.1M sodium borate/boric acid buffer, pH 9.5. A 25 μMsolution of a DNA modified with a 1-n-hexyl amine group in the boratebuffer is placed on the surface of the substrate. The amine groups ofthe DNA bind to the activated MHA molecules forming a DNA dot having a100 nm diameter. The procedure of forming an MHA dot and binding a DNAmolecule to it is repeated many more times with different amine modifiedDNA molecules to form a master having a DNA array with feature of about100 nm.

The master is used to print a complement image array of DNA sequences ona second substrate in which each DNA sequence is complementary to one ofthe DNA molecules on the master and is located at a position on thesecond substrate that is a mirror image of its complementary sequence onthe master. The complement image array is prepared by modifying a set ofDNA molecules that includes all of the DNA molecules that arecomplementary to the DNA molecules on the master with a hexyl thiollinker. The thiol modified DNA molecules are placed in a phosphatebuffer having a pH of 6.8 and 1 M NaCl. The master is immersed in thesolution containing the thiol modified DNA molecules for 2 hrs, thenmaster is removed from the solution and rinsed with 1 M NaCl in TEbuffer once and with water five times.

A second clean gold on mica substrate is placed in contact with themaster so that the two gold surfaces are facing each other and have asmall amount of water in between them. A small mechanical force isapplied to push the two substrates together. As water between the twosubstrates evaporates, the spacing between the surfaces decreases due toincreasing capillary attraction forces. Consequently, thiol groups ofthe thiol modified DNA molecules approach the second substrate and bindto it. After about 5 hrs, the substrates are dipped into 1M NaCl in TEbuffer solution (70° C.) for 20 min. The substrates spontaneouslyseparate and are rinsed with 1M NaCl in TE buffer 2 times and with waterfive times, then allowed to air dry. The master can be used to prepareone or more additional complement images.

Example 4 Preparation of a Complement Image of a DNA Array

A DNA chip is purchased and used as a primary master. The DNA chip has a12×12 square array in which each square is 300 nm×300 nm and has adifferent DNA sequence attached to a substrate for a total of 144different DNA sequences. The 300 nm×300 nm squares are spaced 100 nmapart along the x- and y-axis of the surface of the substrate.

The master is used to print a 12×12 complement image array of DNAsequences on a second substrate in which each DNA sequence iscomplementary to one of the DNA molecules on the master and is locatedat a position on the second substrate that is a mirror image of itscomplementary sequence on the master. A set of DNA molecules thatincludes all of the DNA molecules that are complementary to the DNAmolecules on the master (i.e., 144 different complementary DNAsequences) are modified with a hexyl thiol linker. The thiol modifiedDNA molecules are placed in a phosphate buffer having a pH of 6.8 and 1M NaCl. The master is immersed in the solution containing the thiolmodified DNA molecules for 2 hrs, then master is removed from thesolution and rinsed with 1 M NaCl in TE buffer once and with water fivetimes.

A clean gold on mica substrate is placed in contact with the master sothat the gold surface of the new substrate is facing the 12×12 array ofDNA molecules. A small amount of water is in between the two surfaces. Asmall mechanical force is applied to push the two substrates together.As water between the two substrates evaporates, the spacing between thesurfaces decreases due to increasing capillary attraction forces.Consequently, thiol groups of the thiol modified DNA molecules approachthe second substrate and bind to it. After about 5 hrs, the substratesare dipped into 1M NaCl in TE buffer solution (70° C.) for 20 min. Thesubstrates spontaneously separate and are rinsed with 1M NaCl in TEbuffer 2 times and with water five times, then allowed to air dry. Thecomplement image has a 12×12 array of DNA molecules that arecomplementary to the DNA molecules on the master. The master can be usedto prepare one or more additional complement image arrays following thesame procedure.

In addition, the primary master can be replicated one or more times byfollowing the procedure, as described above, except that the complementimage is used in place of the master and a third set of 144 DNAmolecules having the same sequences as the DNA molecules on the primarymaster and modified with a hexyl thiol linker is assembled on thecomplement image. A third substrate of gold on mica is then brought incontact with the complement image as described above for the primarymaster and the second substrate. The third set of DNA bound to the thirdsubstrate and separated from the complement image is a replica of theprimary master.

Other embodiments of the invention will be apparent to those skilled inthe art from a consideration of the specification or practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with the true scope and spiritof the invention being indicated by the following claims.

1.-110. (canceled)
 111. A composition, comprising: a) a mastercomprising a pattern of a first set of molecules bound to a firstsubstrate; and b) a complement image comprising a pattern of a secondset of molecules bound to a second substrate via a reactive functionalgroup on each molecule of the second set of molecules, wherein eachmolecule in the second set of molecules has a recognition component thatbinds to at least a portion of a molecule from the first set ofmolecule.
 112. The composition of claim 111, wherein each molecule ofthe second set of molecule further comprises one or more of thefollowing components: a) an exposed functionality; b) a covalent bond ora first spacer that links the reactive functional group to therecognition component; and c) a covalent bond or a second spacer thatlinks the exposed functionality to the recognition component.
 113. Thecomposition of claim 112, wherein the second set of molecules comprisestwo or more different molecules.
 114. The composition of claim 113,wherein one or more molecules from the first set of molecules determineswhere each of the molecules from the second set of molecules binds. 115.The composition of claim 114, wherein two or more molecules of thesecond set of molecules have different recognition components.
 116. Thecomposition of claim 114, wherein two or more molecules of the secondset of molecules have both different recognition components anddifferent exposed functionalities.
 117. The composition of claim 114,wherein the two or more different molecules of the second set ofmolecules form a pattern on the second substrate that has a heightprofile that comprises two or more depths.
 118. The composition of claim117, wherein at least one of the two or more different moleculescomprises a first spacer, and another of the two or more differentmolecules either does not comprise a spacer or comprises a second spacerthat has a different length from the first spacer.
 119. The compositionof claim 112, wherein a lateral dimension of at least one feature of thecomplement image is less than 200 nm.
 120. The composition of claim 112,wherein the bonds foraied between the first set of molecules and thesecond set of molecules are hydrogen bonds, ionic bonds, covalent bonds,van der Waals bonds, or a combination thereof.
 121. The composition ofclaim 120, wherein the bonds between the first set of molecules and thesecond set of molecules are broken.
 122. The composition of claim 120,wherein the bonds formed between the first set of molecules and thesecond set of molecules are hydrogen bonds.
 123. The composition ofclaim 112, wherein the reactive functional group on the second set ofmolecules is a thiol group or a protected thiol group, and the surfaceof the second substrate is gold, silver, copper, cadmium, zinc,palladium, platinum, mercury, lead, iron, chromium, manganese, tungsten,or any alloys thereof.
 124. The composition of claim 112, wherein thereactive functional group on the second set of molecules is a silane ora chlorosilane, and the surface of the second substrate is doped orundoped silicon.
 125. The composition of claim 112, wherein the reactivefunctional group on the second set of molecules is a carboxylic acid,and the surface of the second substrate is an oxide.
 126. Thecomposition of claim 125, wherein the oxide is silica, alumina, quartz,or glass.
 127. The composition of claim 112, wherein the reactivefunctional group on the second set of molecules is a nitrile or anisonitrile, and the surface of the second substrate is platinum,palladium or any alloy thereof.
 128. The composition of claim 112,wherein the reactive functional group on the second set of molecules isa hydroxamic acid, and the surface of the second substrate is copper.129. The composition of claim 112, wherein a component of each of thefirst set of molecules is a nucleic acid sequence and the recognitioncomponent of the second set of molecules is a nucleic acid sequence thathas at least three consecutive bases that are complementary to at leastthree consecutive bases of at least one molecule from the first set ofmolecules.
 130. The composition of claim 129, wherein the bonds formedbetween the first set of molecules and the second set of molecules arehydrogen bonds.
 131. The composition of claim 130, wherein the secondset of molecules comprises two or more different molecules,
 132. Thecomposition of claim 131, wherein one or more molecules from the firstset of molecules determines where each of the molecules from the secondset of molecules binds.
 133. The composition of claim 132, wherem thefirst set of molecules comprises two or more molecules having differentnucleic acid sequences.
 134. The composition of claim 132, wherein twoor more molecules of the second set of molecules have different nucleicacid sequences.
 135. The composition of claim 134, wherein the nucleicacid sequence of the first and second sets of molecules are selectedfrom the group consisting of DNA, RNA, modified nucleic acid sequencesand combinations thereof.
 136. The composition of claim 135, wherein thehydrogen bonds between the first set of molecules and the second set ofmolecules are broken.
 137. The composition of claim 112, wherein acomponent of each of the first set of molecules is a peptide nucleicacid (PNA) sequence and the recognition component of the second set ofmolecules is a PNA sequence.
 138. The composition of claim 121, wheremthe exposed functionality of each molecule of the second set ofmolecules is absent or is, independently, selected from the groupconsisting of —OH, —CONH—, —CONHCO—, —NH₂₎—NH—, —COOH, —COOR, —CSNH—,—NO₂″, —SO₂, —SH, —RCOR—, —RCSR—, —RSR, —ROR—, —PO₄″³, —OSO₃″²,—SO₃″_(?)-COO″, —SOO′, —RSOR—, —CONR₂, —(OCH₂CH₂)_(n)OH (where n=1-20,preferably 1-8), —CH₃, —PO₃H″, -2-imidazole, —N(CH₃)₂, —NR₂, —PO3H2,—CN, —(CF₂)RCF₃ (where n=1-20, preferably 1-8), and an olefin, wherein,R is hydrogen, a hydrocarbon, a halogenated hydrocarbon, a protein, anenzyme, a carbohydrates, a lectin, a hormone, a receptor, an antigen, anantibody, or a hapten.
 139. The composition of claim 138, furthercomprising a metal or a metal ion bound to the exposed functional groupof at least one molecule from the second set of molecules.
 140. Thecomposition of claim 139, wherein the exposed functional group is TMSH,and the metal or metal ion is Au°, Ag°, or Ag⁺.
 141. The composition ofclaim 139, wherein the exposed functional group is —COOH, and the metalor metal ion is Ag° or Ag⁺.
 142. The composition of claim 112, whereineach molecule of the second set of molecules has a first spacer, asecond spacer, or a first and the second spacer, and the spacers are,independently, selected from the group consisting of an alkylene, aheteroalkylene, a heterocycloalkylene, an alkenylene, an alkynylene, anarylene, a heteroarylene, arylalkylene, and a heteroarylalkylene,wherein the alkylene, heteroalkylene, heterocycloalkylene, alkenylene,alkynylene, arylene, heteroarylene, arylalkylene, or heteroarylalkylenemay be substituted or unsubstituted.
 143. The composition of claim 142,wherein the substituents for the alkylene, a heteroalkylene, analkenylene, an alkynylene, an arylene, a heteroarylene, aheterocycloalkylene, an arylalkylene, and a heteroarylalkylene areselected from the group consisting of halogens and hydroxy.
 144. Thecomposition of claim 121, wherein at least one portion of the secondsubstrate surface is free of the second set of molecules.
 145. Acomposition, comprising: a) a first pattern of a first set of moleculesbound to a first substrate; and b) a second pattern of a second set ofmolecules bound to a second substrate via a reactive functional group oneach molecule of the second set of molecules, wherein each molecule ofthe second set of molecules comprises a recognition component that bindsto at least one molecule in the first set of molecules, and wherein thesecond pattern is a complement image of the first pattern.
 146. Thecomposition of claim 145, wherein each molecule of the second set ofmolecule further comprises one or more of the following components: a)an exposed functionality; b) a covalent bond or a first spacer thatlinks the reactive functional group to the recognition component; and c)a covalent bond or a second spacer that links the exposed functionalityto the recognition component.
 147. The composition of claim 146, whereinthe second set of molecules comprises two or more different molecules.148. The composition of claim 147, wherein one or more molecules fromthe first set of molecules determines where each of the molecules fromthe second set of molecules binds.
 149. The composition of claim 148,wherein two or more molecules of the second set of molecules havedifferent recognition components.
 150. The composition of claim 148,wherein two or more molecules of the second set of molecules have bothdifferent recognition components and different exposed functionalities.151. The composition of claim 148, wherein the two or more differentmolecules of the second set of molecules form a pattern on the secondsubstrate that has a height profile that comprises two or more depths.152. The composition of claim 151, wherein at least one of the two ormore different molecules comprises a first spacer, and another of thetwo or more different molecules either does not comprise a spacer orcomprises a second spacer that has a different length from the firstspacer.
 153. The composition of claim 146, wherein a lateral dimensionof at least one feature of the complement image is less than 200 nm.154. The composition of claim 146, wherein the reactive functional groupof the second set of molecules is a thiol group and the surface of thesecond substrate is gold, silver, copper, cadmium, zinc, palladium,platinum, mercury, lead, iron, chromium, manganese, tungsten, or anyalloys thereof.
 155. The composition of claim 146, wherein the reactivefunctional group of the second set of molecules is a silane or achlorosilane group and the surface of the second substrate is doped orundoped silicon.
 156. The composition of claim 146, wherein the reactivefunctional group of the second set of molecules is a carboxylic acid,and the surface of the second substrate is an oxide.
 157. Thecomposition of claim 156, wherein the oxide is silica, alumina, quartz,or glass.
 158. The composition of claim 146, wherein the reactivefunctional group of the second monolayer of complementary molecules is anitrile or an isonitrile group, and the surface of the second substrateis platinum, palladium or any alloy thereof.
 159. The composition ofclaim 146, wherein the reactive functional group of the second monolayerof complementary molecules is a hydroxamic acid, and the surface of thesecond substrate is copper.
 160. The composition of claim 146, wherein acomponent of each of the first set of molecules is a nucleic acidsequence and the recognition component of the second set of molecules isa nucleic acid sequence that has at least three consecutive bases thatare complementary to at least three consecutive bases of at least onemolecule from the first set of molecules.
 161. The composition of claim160, wherein the second set of molecules comprises two or more differentmolecules.
 162. The composition of claim 161, wherein one or moremolecules from the first set of molecules determines where each of themolecules from the second set of molecules binds.
 163. The compositionof claim 162, wherein the first set of molecules comprises two or moremolecules having different nucleic acid sequences.
 164. The compositionof claim 162, wherein two or more molecules of the second set ofmolecules have different nucleic acid sequences.
 165. The composition ofclaim 164, wherein the nucleic acid sequence of the first and secondsets of molecules are selected from the group consisting of DNA, RNA,modified nucleic acid sequences and combinations thereof.
 166. Thecomposition of claim 146, wherein a component of each of the first setof molecules is a peptide nucleic acid (PNA) sequence and therecognition component of the second set of molecules is a PNA sequence.167. The composition of claim 146, wherein the exposed functionality ofeach molecule of the second set of molecules is absent or is,independently, selected from the group consisting of —OH, —CONH—,—CONHCO—, —NH₂, —NH—, —COOH, —COOR, —CSNH—, —NO₂″, —SO₂, —SH, —RCOR—,—RCSR—, —RSR, —ROR—, —PO₄″³, —OSO₃″², —SO₃″, —COO″, —SOO″, —RSOR—,—CONR₂, —(OCH₂CH₂)_(n)OH (where n=1-20, preferably 1-8), —CH₃, —PO₃H*,-2-imidazole, —N(CH₃)₂, —NR₂, —PO₃H₂, —CN, —(CF₂)_(n)CF₃ (where n=1-20,preferably 1-8), and an olefin, wherein, R is hydrogen, a hydrocarbon, ahalogenated hydrocarbon, a protein, an enzyme, a carbohydrates, alectin, a hormone, a receptor, an antigen, an antibody, or a hapten.168. The composition of claim 167, further comprising a metal or a metalion bound to the exposed functional group of at least one molecule fromthe second set of molecules.
 169. The composition of claim 168, whereinthe exposed functional group is -˜SH, and the metal or metal ion is Au°,Ag°, or Ag⁺.
 170. The composition of claim 168, wherein the exposedfunctional group is —COOH, and the metal or metal ion is Ag° or Ag⁺.171. The composition of claim 146, wherein each molecule of the secondset of molecules has a first spacer, a second spacer, or a first and thesecond spacer, and the spacers are, independently, selected from thegroup consisting of an alkylene, a heteroalkylene, aheterocycloalkylene, an alkenylene, an alkynylene, an arylene, aheteroarylene, arylalkylene, and a heteroarylalkylene, wherein thealkylene, heteroalkylene, heterocycloalkylene, alkenylene, alkynylene,arylene, heteroarylene, arylalkylene, or heteroarylalkylene may besubstituted or unsubstituted.
 172. The composition of claim 171, whereinthe substituents for the alkylene, a heteroalkylene, aheterocycloalkylene, an alkenylene, an alkynylene, an arylene, aheteroarylene, an arylalkylene, and a heteroarylalkylene are selectedfrom the group consisting of halogens and hydroxy.
 173. The compositionof claim 146, wherein at least one portion of the second substratesurface is free of the second set of molecule.
 174. The composition ofclaim 146, wherein the portion of the second substrate that is free ofthe second set of molecules has been degraded.
 175. The composition ofclaim 173, wherein a material has been deposited on the portion of thesecond substrate that is free of the second set of molecules.
 176. Thecomposition of claim 175, wherein the deposited material is selectedfrom the group consisting of semiconductors, dielectrics, metals, metaloxides, metal nitrides, metal carbides, and combinations thereof.
 177. Acomposition, comprising: a) a first pattern of a first set of moleculesbound to a first substrate; and b) a second substrate, wherein thesecond substrate comprises a degraded portion and an undegraded portionand is a complement image of the first pattern.
 178. The composition ofclaim 177, wherein the complement image is formed by a method comprisingthe steps of: a) forming a second pattern of a second set of moleculeson the second substrate, wherein the second pattern is a complementimage of the first pattern, and wherein at least one portion of thesecond substrate surface is free of the second set of molecules; b)degrading the portion of the second substrate that is free of the secondset of molecules; and c) removing the second set of molecules from thesecond substrate, thereby exposing the surface of the second substrate.179. A composition, comprising: a) a first pattern of a first set ofmolecules bound to a first substrate; and b) a second substrate having apatterned layer of a material deposited thereon, wherein the patternedlayer of deposited material on the second substrate is a complementimage of the first pattern.
 180. The composition of claim 179, whereinthe complement image is formed on the second substrate by a methodcomprising the steps of; a) forming a second pattern of a second set ofmolecules on the second substrate, wherein the second pattern is acomplement image of the first pattern, and wherein at least one portionof the second substrate surface is free of the second set of molecules;b) depositing a material on the portion of the second substrate that isfree of the second set of molecules; and c) removing the second set ofmolecules from the second substrate, thereby exposing the surface of thesecond substrate.
 181. The composition of claim 179, wherein thedeposited material is selected from the group consisting ofsemiconductors, dielectrics, metals, metal oxides, metal nitrides, metalcarbides, and combinations thereof. 182.-245. (canceled)